Fragrance and methods for production of 5-epi-β-vetivone, 2-isopropyl-6,10-dimethyl-spiro[4.5]deca-2,6-dien-8-one, and 2-isopropyl-6,10-dimethyl-spiro[4.5]deca-1,6-dien-8-one

ABSTRACT

The present invention is directed to novel methods for production of 5-epi-β-vetivone, 2-isopropyl-6,10-dimethyl-spiro[4.5]deca-2,6-dien-8-one and 2-isopropyl-6,10-dimethyl-spiro[4.5]deca-1,6-dien-8-one, which are useful for their fragrant qualities. In one embodiment the present invention describes a method for production of 5-epi-β-vetivone by the use of premnaspirodiene as starting material. In another embodiment the present invention describes a method for production of 2-isopropyl-6,10-dimethyl-spiro[4.5]deca-2,6-dien-8-one and 2-isopropyl-6,10-dimethyl-spiro[4.5]deca-1,6-dien-8-one by the use of premnaspirodiene as starting material. In yet another embodiment the present invention describes a novel method for production of premnaspirodiene from a terpene substrate. Use of the fragrant components or any composition containing the component can be advantageously employed in the perfumery industry.

CROSS-REFERENCES

This application is a continuation in part of U.S. patent applicationSer. No. 12/052,464, filed Mar. 20, 2008, which claims priority fromU.S. Provisional Application Ser. No. 60/919,284 filed on Mar. 20, 2007,which are both incorporated herein in their entirety by reference.

FIELD OF THE INVENTION

The present invention is directed to novel methods for production of5-epi-β-vetivone,2-isopropyl-6,10-dimethyl-spiro[4.5]deca-2,6-dien-8-one and2-isopropyl-6,10-dimethyl-spiro[4.5]deca-1,6-dien-8-one, which areuseful for their fragrant qualities. In one embodiment the presentinvention describes a method for production of 5-epi-β-vetivone by theuse of premnaspirodiene as starting material. In another embodiment thepresent invention describes a method for production of2-isopropyl-6,10-dimethyl-spiro[4.5]deca-2,6-dien-8-one and2-isopropyl-6,10-dimethyl-spiro[4.5]deca-1,6-dien-8-one by the use ofpremnaspirodiene as starting material. In yet another embodiment thepresent invention describes a novel method for production ofpremnaspirodiene from a terpene substrate. Use of the fragrantcomponents or any composition containing the component can beadvantageously employed in the perfumery, personal care and consumerproducts industries.

BACKGROUND OF THE INVENTION

The present invention relates to the field of fragrance compounds andcompositions. In particular, the invention concerns the method ofproduction of fragrance compositions as well as their use as, e.g.,perfuming, personal care and consumer product ingredients.

5-epi-β-vetivone, represented by Formula (1) below:

is a potentially valuable fragrance component or perfuming ingredientdue to its vetivert, woody, grapefruit aroma. Similarly, the mixture of2-isopropyl-6,10-dimethyl-spiro[4.5]deca-2,6-dien-8-one, represented byFormula (3) below

and 2-isopropyl-6,10-dimethyl-spiro[4.5]deca-1,6-dien-8-one, representedby Formula (4) below

is a potentially valuable fragrance component as well, due to itsvetivert, woody, grapefruit aroma.

One method of producing 5-epi-β-vetivone has been developed to date(11). In this process 5-epi-β-vetivone was made as a byproduct duringthe diastereoselective 1,4-addition of a methyl group to a double bondto synthesize (−)-solavetivone (5). However, 5-epi-β-vetivone is made asa byproduct and the disclosed process is not suitable for commercialgrade production of 5-epi-β-vetivone.

No methods of producing2-isopropyl-6,10-dimethyl-spiro[4.5]deca-2,6-dien-8-one (3) and2-isopropyl-6,10-dimethyl-spiro[4.5]deca-1,6-dien-8-one (4) are known todate.

Therefore, there is a current need in the art for methods to produce5-epi-β-vetivone (1) in a fewer number of reaction steps, with higheroverall yield, and from less expensive and more available startingmaterials. Furthermore, there is a current need in the art for methodsto produce 2-isopropyl-6,10-dimethyl-spiro[4.5]deca-2,6-dien-8-one (3)and 2-isopropyl-6,10-dimethyl-spiro[4.5]deca-1,6-dien-8-one (4).

Accordingly, the present invention is directed to novel methods forproduction of 5-epi-β-vetivone,2-isopropyl-6,10-dimethyl-spiro[4.5]deca-2,6-dien-8-one (3) and2-isopropyl-6,10-dimethyl-spiro[4.5]deca-1,6-dien-8-one (4), which areuseful for their fragrant qualities. The production of the fragrancecompositions as described by the present invention can be used for thepreparation of perfumes, household products, laundry products, body careproducts or cosmetic products, as well as related compositions andarticles. Mixtures of the compound described in the invention, afragrance modifying composition, a perfume composition and a colognecomposition are also included in the invention. The compounds andcompositions can be employed in order to confer a woody, grapefruit, orvetivert odor to a variety of products.

SUMMARY OF THE INVENTION

In one embodiment the present invention describes a method forsynthesizing 5-epi-β-vetivone (1) comprising the steps of:

(1) subjecting (−)-premnaspirodiene (2) to oxidation of an allyliccarbon to form (−)-solavetivone; and

(2) subjecting the (−)-solavetivone (5) formed in step (1) toacid-catalyzed isomerization to form 5-epi-β-vetivone (1);

wherein time and temperature conditions for step (2) are such that thepredominant product is 5-epi-β-vetivone (1).

In another embodiment the present invention describes a method forsynthesizing a mixture of2-isopropyl-6,10-dimethyl-spiro[4.5]deca-2,6-dien-8-one (3) and2-isopropyl-6,10-dimethyl-spiro[4.5]deca-1,6-dien-8-one (4) comprisingthe steps of:

(1) subjecting (−)-premnaspirodiene (2) to oxidation of an allyliccarbon to form (−)-solavetivone (5); and

(2) subjecting the (−)-solavetivone (5) formed in step (1) toacid-catalyzed isomerization to form the mixture of2-isopropyl-6,10-dimethyl-spiro[4.5]deca-2,6-dien-8-one (3) and2-isopropyl-6,10-dimethyl-spiro[4.5]deca-1,6-dien-8-one (4);

wherein time and temperature conditions for step (2) are such that thepredominant product is a mixture of2-isopropyl-6,10-dimethyl-spiro[4.5]deca-2,6-dien-8-one (3) and2-isopropyl-6,10-dimethyl-spiro[4.5]deca-1,6-dien-8-one (4).

In another embodiment the present invention describes a substantiallypurified and isolated mixture of2-isopropyl-6,10-dimethyl-spiro[4.5]deca-2,6-dien-8-one (3) and2-isopropyl-6,10-dimethyl-spiro[4.5]deca-1,6-dien-8-one (4).

In another embodiment the present invention describes the substantiallypurified and isolated compound2-isopropyl-6,10-dimethyl-spiro[4.5]deca-2,6-dien-8-one (3),substantially free of2-isopropyl-6,10-dimethyl-spiro[4.5]deca-1,6-dien-8-one (4).

In another embodiment the present invention describes the substantiallypurified and isolated compound2-isopropyl-6,10-dimethyl-spiro[4.5]deca-1,6-dien-8-one (3),substantially free of2-isopropyl-6,10-dimethyl-spiro[4.5]deca-2,6-dien-8-one (4).

In another embodiment the present invention describes a method forproducing (−)-premnaspirodiene (2) from a terpene substrate, comprisingthe steps of:

(1) providing the terpene substrate to a host cell transformed ortransfected with a vector comprising a sequence encoding a Hyoscyamusmuticus premnaspirodiene synthase gene;

(2) culturing the host cell under conditions suitable to produce(−)-premnaspirodiene (2) from the terpene substrate; and

(3) isolating the (−)-premnaspirodiene (2).

In another embodiment the present invention describes a method forsynthesizing 5-epi-β-vetivone comprising the steps of:

(1) providing a terpene substrate to a host cell transformed ortransfected with a vector comprising a sequence encoding a Hyoscyamusmuticus premnaspirodiene synthase gene;

(2) culturing the host cell under conditions suitable to produce(−)-premnaspirodiene from the terpene substrate; and

(3) isolating the (−)-premnaspirodiene;

(4) subjecting the isolated (−)-premnaspirodiene to oxidation of anallylic carbon to form (−)-solavetivone; and

(5) subjecting the (−)-solavetivone formed in step (4) to acid-catalyzedisomerization to form 5-epi-β-vetivone;

wherein time and temperature conditions for step (5) are such that thepredominant product is 5-epi-β-vetivone.

In another embodiment the present invention describes a method forsynthesizing a mixture of2-isopropyl-6,10-dimethyl-spiro[4.5]deca-2,6-dien-8-one and2-isopropyl-6,10-dimethyl-spiro[4.5]deca-1,6-dien-8-one comprising thesteps of:

(1) providing a terpene substrate to a host cell transformed ortransfected with a vector comprising a sequence encoding a Hyoscyamusmuticus premnaspirodiene synthase gene;

(2) culturing the host cell under conditions suitable to produce(−)-premnaspirodiene from the terpene substrate; and

(3) isolating the (−)-premnaspirodiene;

(4) subjecting the isolated (−)-premnaspirodiene to oxidation of anallylic carbon to form (−)-solavetivone; and

(5) subjecting the (−)-solavetivone formed in step (4) to acid-catalyzedisomerization to form the mixture of2-isopropyl-6,10-dimethyl-spiro[4.5]deca-2,6-dien-8-one and2-isopropyl-6,10-dimethyl-spiro[4.5]deca-1,6-dien-8-one;

wherein time and temperature conditions for step (5) are such that thepredominant product is the mixture of2-isopropyl-6,10-dimethyl-spiro[4.5]deca-2,6-dien-8-one and2-isopropyl-6,10-dimethyl-spiro[4.5]deca-1,6-dien-8-one.

In yet another embodiment the present invention describes a method forsynthesizing 2-isopropyl-6,10-dimethyl-spiro[4.5]deca-2,6-dien-8-onecomprising the steps of:

(1) providing a terpene substrate to a host cell transformed ortransfected with a vector comprising a sequence encoding a Hyoscyamusmuticus premnaspirodiene synthase gene;

(2) culturing the host cell under conditions suitable to produce(−)-premnaspirodiene from the terpene substrate; and

(3) isolating the (−)-premnaspirodiene;

(4) subjecting the isolated (−)-premnaspirodiene to oxidation of anallylic carbon to form (−)-solavetivone; and

(5) subjecting the (−)-solavetivone formed in step (4) to acid-catalyzedisomerization to form a mixture of2-isopropyl-6,10-dimethyl-spiro[4.5]deca-2,6-dien-8-one and2-isopropyl-6,10-dimethyl-spiro[4.5]deca-1,6-dien-8-one; wherein timeand temperature conditions for step (5) are such that the predominantproduct is the mixture of2-isopropyl-6,10-dimethyl-spiro[4.5]deca-2,6-dien-8-one and2-isopropyl-6,10-dimethyl-spiro[4.5]deca-1,6-dien-8-one; and

(6) isolating the2-isopropyl-6,10-dimethyl-spiro[4.5]deca-2,6-dien-8-one from the mixtureof 2-isopropyl-6,10-dimethyl-spiro[4.5]deca-2,6-dien-8-one and2-isopropyl-6,10-dimethyl-spiro[4.5]deca-1,6-dien-8-one formed in step(5) by a method selected from the group consisting of thin-layerchromatography, column chromatography, gas chromatography, andcountercurrent distribution.

In yet another embodiment the present invention describes a method forsynthesizing 2-isopropyl-6,10-dimethyl-spiro[4.5]deca-1,6-dien-8-onecomprising the steps of:

(1) providing a terpene substrate to a host cell transformed ortransfected with a vector comprising a sequence encoding a Hyoscyamusmuticus premnaspirodiene synthase gene;

(2) culturing the host cell under conditions suitable to produce(−)-premnaspirodiene from the terpene substrate; and

(3) isolating the (−)-premnaspirodiene;

(4) subjecting the isolated (−)-premnaspirodiene to oxidation of anallylic carbon to form (−)-solavetivone; and

(5) subjecting the (−)-solavetivone formed in step (4) to acid-catalyzedisomerization to form a mixture of2-isopropyl-6,10-dimethyl-spiro[4.5]deca-2,6-dien-8-one and2-isopropyl-6,10-dimethyl-spiro[4.5]deca-1,6-dien-8-one; wherein timeand temperature conditions for step (5) are such that the predominantproduct is the mixture of 2-isopropyl-6,10[-dimethyl-spiro4.5]deca-2,6-dien-8-one and2-isopropyl-6,10-dimethyl-spiro[4.5]deca-1,6-dien-8-one; and

(6) isolating the2-isopropyl-6,10-dimethyl-spiro[4.5]deca-1,6-dien-8-one from the mixtureof 2-isopropyl-6,10-dimethyl-spiro[4.5]deca-2,6-dien-8-one and2-isopropyl-6,10-dimethyl-spiro[4.5]deca-1,6-dien-8-one formed in step(e) by a method selected from the group consisting of thin-layerchromatography, column chromatography, gas chromatography, andcountercurrent distribution. The present invention also includes afragrance composition with a compound having the structure:

in an amount effective to impart a fragrance. In one aspect, thecompound of the fragrance composition is present in an amount of atleast 0.01% by weight. In another aspect, the fragrance compositioncomprises an amount of the compound of Formula (1) effective to impartfragrance in combination with conventional fragrance ingredients.

Another object of the present invention is a perfumed product comprisinga compound having the structure of Formula (1). The perfumed product canbe a household product, such as, for example, a solid or liquiddetergent, a fabric softener, an air freshener, a fabric refresher, anironing water, a paper, a wipe or a bleach. The perfumed product can bea cosmetic product or a body care product, for example a perfume, acologne or after-shave lotion, a perfumed soap, a shower or bath salt,mousse, oil or gel, a hygiene product, a hair care product, a shampoo, adeodorant or antiperspirant.

The present invention can be a perfuming composition containing thecompound of Formula (1) in an amount sufficient to give a fragrance tothe composition. In one embodiment, the perfuming composition canadditionally contain at least one perfumery adjuvant.

Another aspect of the present invention is a fragrance applicationcomprising a compound of Formula (1). The fragrance application can be,for example, a household product such as laundry product, a solid orliquid detergent, a fabric softener, an air freshener, a fabricrefresher, an ironing water, a paper, a wipe or a bleach. In anotheraspect, the fragrance application can be a cosmetic product or body careproduct such as a perfume, a cologne or after-shave lotion, a perfumedsoap, a shower or bath salt, mousse, oil or gel, a hygiene product, ahair care product, a shampoo, a deodorant or antiperspirant. A consumerproduct is a product which is compatible with perfuming ingredients. Inother words, a perfumed article according to the invention comprises thefunctional formulation, as well as optionally additional benefit agents,corresponding to a consumer product, e.g. a detergent or an airfreshener, and an olfactive effective amount of at least one compound.

The invention also includes methods of imparting a woody, vetivert orgrapefruit odor to a fragrance or fragrance composition by providing thecompound of Formula (1) to the fragrance.

Methods of the invention include imparting, improving, enhancing ormodifying a fragrance formulation through the addition of an olfactoryacceptable amount of Formula (1). The olfactory acceptable amount can befrom about 0.005 to about 10 weight percent of the fragranceformulation. In one aspect, the olfactory acceptable amount is fromabout 0.5 to about 8 weight percent of the fragrance formulation. Inanother aspect, the method of claim 18, wherein the olfactory acceptableamount is from about 1 to about 7 weight percent of the fragranceformulation.

The invention also includes a mixture comprising the compound of Formula(1) and an auxiliary ingredient compatible with the compound of Formula(1), the weight ratio of the compound of Formula (1) being in the rangeof from about 1:1 up to about 1:5.

The invention also includes a fragrance modifying composition comprisingthe compound of Formula (1) and an auxiliary ingredient compatible withthe compound of Formula (1), the weight ratio of the compound of Formula(1) being in the range of from about 1:1 up to about 1:5.

Also included in the invention is a perfume composition comprising thecompound of Formula (1) and at least one compatible adjuvant, the ratioof the compound of Formula (1) being in the range of from about 1:1 upto about 1:5.

The invention also includes a cologne composition comprising thecompound of Formula (1) and at least one compatible adjuvant, the ratioof the compound of Formula (1) being in the range of from about 1:1 upto about 1:5. The invention also includes a method of using the compoundof Formula (1) as a perfume. In one embodiment, the method of using thecompound of Formula (1) as a perfume further comprises an auxiliaryingredient compatible with the compound of Formula (1).

Other features and advantages of the invention will become apparent fromthe following description of the preferred embodiments thereof and fromthe claims.

BRIEF DESCRIPTION OF THE DRAWINGS

The following invention will become better understood with reference tothe specification, appended claims, and accompanying drawings, where:

FIG. 1 depicts the structural formula of 5-epi-β-vetivone (1).

FIG. 2 depicts the structural formula of premnaspirodiene (2).

FIG. 3 depicts the structural formulas of2-isopropyl-6,10-dimethyl-spiro[4.5]deca-2,6-dien-8-one (3) (the“2,6-diene isomer”) and2-isopropyl-6,10-dimethyl-spiro[4.5]deca-1,6-dien-8-one (4) (the“1,6-diene isomer”). FIG. 3A depicts the structural formula of the2,6-diene isomer. FIG. 3B depicts the structural formula of the1,6-diene isomer.

FIG. 4 provides a schematic representation of an oxidation and acidcatalyzed isomerization reaction used in the production methods of thepresent invention.

FIG. 5 provides a schematic representation of an oxidation and acidcatalyzed isomerization reaction used in the production of5-epi-β-vetivone (1).

FIG. 6 provides a schematic representation of an oxidation and acidcatalyzed isomerization reaction used in the production of2-isopropyl-6,10-dimethyl-spiro[4.5]deca-2,6-dien-8-one (3) and2-isopropyl-6,10-dimethyl-spiro[4.5]deca-1,6-dien-8-one (4).

FIG. 7 provides a graph showing the GC trace of premnaspirodiene invegetable oil before distillation (Example 8). FIG. 7A shows the graphon a normal scale. FIG. 7B expands the scale to depict the impuritiespresent in the premnaspirodiene before distillation.

FIG. 8 provides a graph showing the GC trace of purifiedpremnaspirodiene after distillation (Example 8). FIG. 8A shows the graphon a normal scale. FIG. 8B expands the scale to depict the lack ofimpurities present in the premnaspirodiene after distillation.

DETAILED DESCRIPTION OF THE INVENTION

5-epi-β-vetivone (1) is commercially useful in the fragrance industrydue to its vetivert, woody, grapefruit aroma. The present inventiongenerally describes a method for production of2-isopropylidene-6,10-dimethyl-spiro[4.5]dec-6-en-8-one (the“5-epi-β-vetivone”), represented by Formula (1), from (−)premnaspirodiene, represented by Formula (2) below:

In general, one embodiment of the present invention is a method forsynthesizing 5-epi-β-vetivone comprising the steps of:

(1) subjecting (−)-premnaspirodiene (2) to oxidation of an allyliccarbon to form (−)-solavetivone; and

(2) subjecting the (−)-solavetivone (5) formed in step (i) toacid-catalyzed isomerization to form 5-epi-β-vetivone (1);

wherein time and temperature conditions for step (ii) are such that thepredominant product is 5-epi-β-vetivone.

Typically, the allylic carbon oxidation step can be carried outaccording to a scheme demonstrated in FIG. 5 and described in literature[Hwu, J. R.; Wetzel, J. M., J. Org. Chem. (1992), 57(3), 922-928].Furthermore, the acid catalyzed isomerization step can be carried outaccording to Scheme (2) below:

The allylic oxidation reaction step may be carried out using metaloxidants, wherein such oxidants include, but are not limited to,chromium, copper, rhodium, cobalt, manganese, or vanadium. Amongst thechromium oxidants, one may use CrO₃-pyridine complex, chromium trioxideand 3,5-dimethylpyrazole, chromic acid (CrO₂Cl₂) and3,5-dimethylpyrazole, pyridinium chlorochromate (PCC), pyridiniumdichromate (PDC), sodium chromate, sodium dichromate in acetic acid,pyridinium fluorochromate, or 3,5-dimethylpyrazolium fluorochromate(VI).Amongst the copper oxidants, one may use copper(I) bromide, copper(I)iodide, or Cu(OAc)₂ and t-BuOOH. Amongst the rhodium oxidants, one mayuse dirhodium catalysts such as dirhodium tetrakiscaprolactamate(Rh₂(cap)₄), Rh₂(OAc)₄ or dirhodium tetrakisperfluorobutanoateRh₂(pfb)₄). Amongst the cobalt oxidants, one may use Co(OAc)₂ andt-BuOOH. Amongst the manganese oxidants, one may use Mn(OAc)₂ andt-BuOOH. Amongst the vanadium oxidants, one may use V(OAc)₂ and t-BuOOH.Other, alternative, oxidants are described below.

In a preferred embodiment, the metal oxidant is a chromium or rhodiumcatalyst such as CrO₃-pyridine complex, chromium trioxide and3,5-dimethylpyrazole, chromic acid (CrO₂Cl₂) and 3,5-dimethylpyrazole,pyridinium chlorochromate (PCC), pyridinium dichromate (PDC), sodiumchromate, sodium dichromate in acetic acid, pyridinium fluorochromate,3,5-dimethylpyrazolium fluorochromate(VI), Rh₂(cap)₄, Rh₂(OAc)₄ orRh₂(pfb)₄.

In a more preferred embodiment, the metal oxidant is a chromium catalystsuch as chromic acid (CrO₂Cl₂) and 3,5-dimethylpyrazole.

In a preferred embodiment, oxidation of the allylic carbon is performedin a solvent. Typically, the solvent comprises at least one solventselected from the group consisting of aliphatic alcohols, ethercompounds, hydrocarbons, halohydrocarbons, and mixtures thereof.

As used herein, the term “aliphatic alcohol” means a straight-chain orbranched-chain aliphatic alcohol of from 1 to 12 carbon atoms, typicallyof from 1 to 6 carbon atoms. As used herein, the term “ether compound”means a linear or cyclic ether of from 4 to 12 carbon atoms. As usedherein, the term “hydrocarbon” means an aliphatic or aromatichydrocarbon of from 5 to 12 carbon atoms wherein the aliphatichydrocarbon is a straight-chain or branched-chain alkane. As usedherein, the term “halohydrocarbon” means an aliphatic or aromatichalohydrocarbon of from 5 to 12 carbon atoms wherein, when thehalohydrocarbon is an aliphatic halohydrocarbon, the hydrocarbon moietyof the halohydrocarbon is a straight-chain or branched-chain alkane, andwherein the halohydrocarbon comprises from 1 to 4 halogens.

In one alternative, the solvent comprises an aliphatic alcohol.Typically, the aliphatic alcohol is selected from the group consistingof methanol, ethanol, n-propanol, and t-butanol. In another alternative,the solvent comprises an ether compound. Typically, the ether compoundis selected from the group consisting of ethyl ether, tetrahydrofuran,and dioxane.

In another alternative, the solvent comprises a hydrocarbon. Typically,the hydrocarbon is selected from the group consisting of pentane,hexane, benzene, and toluene.

In another alternative, the solvent comprises a halohydrocarbon.Preferably, the halohydrocarbon is dichloromethane.

Preferably, oxidation of the allylic carbon is performed in a solventthat is a mixture of t-butanol and dichloromethane.

In another alternative, oxidation of the allylic carbon is performedusing CrO₂Cl₂ as an oxidizing agent and in the presence of3,5-dimethylpyrazole as a catalyst.

In still another preferred alternative, oxidation of the allylic carbonis performed using t-butyl hydroperoxide and sodium chlorite. Typically,in this alternative, the oxidation is performed in a solvent that is amixture of acetonitrile, water, and t-butanol; preferably, the mixtureof acetonitrile, water, and t-butanol is a 3:1:1 mixture ofacetonitrile, water, and t-butanol. In this alternative, preferably, the(−)-premnaspirodiene is first dispersed in a solvent that includes theacetonitrile and water, but not the t-butanol; the t-butyl hydroperoxideis then added, and the reaction heated to 50° C. for 18 h. At thispoint, the t-butanol is added to the reaction, and the reaction isheated for an additional 16 h at 50° C. (Example 7). The reaction iscooled, poured into a 10% solution of sodium sulfite, and extracted withdiethyl ether; the organic layer, containing the desired product, iswashed with saturated sodium bicarbonate, dried over sodium sulfate, andthen adsorbed onto silica gel. The oxidized product is then purified ona silica gel column and eluted first with 5% ethyl acetate in heptaneand then with 10% ethyl acetate in heptane. Details of this procedureare provided in Example 7.

Typically, the acid-catalyzed isomerization step is performed with anacidic isomerization agent. In one alternative, the acidic isomerizationagent is selected from the group consisting of mineral acids, organicprotonic acids, and Lewis acids. Suitable Lewis acids include, but arenot limited to, trifluoroboronetherate, tin chloride, and titaniumtetrachloride. Suitable mineral acids include, but are not limited to,phosphoric acid, sulfuric acid, perchloric acid, hydrohalide acids, andheteropolyacids. A suitable heteropolyacid is H₃[P(W₃O₁₀)₄]. Suitablehydrohalide acids include, but are not limited to, hydrogen chloride,hydrogen bromide, and hydrogen iodide. Suitable organic protonic acidsinclude, but are not limited to, trifluoroacetic acid, acetic acid, andmethylsulfonic acid. In one alternative the acidic isomerization agentis a mixture of acetic acid and sulfuric acid.

In the preferred embodiment, the acidic isomerization agent is a solidphase acidic isomerization agent. Solid phase acidic isomerizationagents within the scope of the present invention include, but are notlimited to, montmorillonite clay, acidic resins, and acidic aluminumoxide. Preferably, the acidic isomerization agent is an acidic resin.Suitable acidic resins include, but are not limited to, Dowex®50(available from Dow Chemical Co.), Amberlyst® IR-15 (available from Rohmand Haas Co.), and Nafion® perfluorinated resin (available from E.I. duPont de Nemours & Co., Inc.).

An appropriate range of reaction time and reaction temperature isnecessary in order to produce 5-epi-β-vetivone (1) from premnaspirodiene(2) according to Scheme (1) below:

In Scheme (1), the first step, the oxidation of (−)-premnaspirodiene to(−)-solavetivone at the allylic carbon, can be performed by any of theoxidation procedures described above, using metal oxidants or,alternatively, using t-butyl hydroperoxide and sodium chlorite.

A reaction time that is too short will result in low yield of5-epi-β-vetivone, since not all of the premnaspirodiene will beconverted to the desired end-product, 5-epi-β-vetivone. A reaction timethat is too long will drive the reaction toward production of a mixtureof the 2,6 diene and 1,6 diene isomers as the end-product and no5-epi-β-vetivone will be produced. Typically, the reaction timepreferred for production of 5-epi-β-vetivone is between about 15 minutesand about 120 minutes and the reaction temperature is between about 25degrees Celsius and about 150 degrees Celsius. More preferably, thereaction time is between about 30 minutes and about 90 minutes. Mostpreferably, the reaction time is between about 40 minutes and about 60minutes. Appropriate reaction temperature is similarly important. In thepreferred embodiment, the reaction temperature is between about 25degrees Celsius and about 150 degrees Celsius. More preferably, thereaction temperature is between about 50 degrees Celsius and about 125degrees Celsius. Most preferably, the reaction temperature is betweenabout 75 degrees Celsius and about 105 degrees Celsius.

In another embodiment the present invention describes a method forsynthesizing a mixture of2-isopropyl-6,10-dimethyl-spiro[4.5]deca-2,6-dien-8-one (3) and2-isopropyl-6,10-dimethyl-spiro[4.5]deca-1,6-dien-8-one (4) comprisingthe steps of:

(1) subjecting (−)-premnaspirodiene (2) to oxidation of an allyliccarbon to form (−)-solavetivone (5); and

(2) subjecting the (−)-solavetivone (5) formed in step (1) toacid-catalyzed isomerization to form the mixture of2-isopropyl-6,10-dimethyl-spiro[4.5]deca-2,6-dien-8-one (3) and2-isopropyl-6,10-dimethyl-spiro[4.5]deca-1,6-dien-8-one (4);

wherein time and temperature conditions for step (ii) are such that thepredominant product is a mixture of2-isopropyl-6,10-dimethyl-spiro[4.5]deca-2,6-dien-8-one (3) and2-isopropyl-6,10[-dimethyl-spiro 4.5]deca-1,6-dien-8-one (4).

Typically, the allylic carbon oxidation step can be carried outaccording to a scheme demonstrated in FIG. 6 and described in literature[Hwu, J. R.; Wetzel, J. M., J. Org. Chem. (1992), 57(3), 922-928].Furthermore, the acid catalyzed isomerization step can be carried outaccording to Scheme (3) below:

Typically, oxidation of the allylic carbon is performed using CrO₂Cl₂ asan oxidizing agent. In the preferred embodiment, the oxidation step isperformed in a solvent. Preferably, the oxidation step is performed in asolvent comprising at least one solvent selected from the groupconsisting of aliphatic alcohols, ether compounds, hydrocarbons,halohydrocarbons, and mixtures thereof. Alternatively, a solventcomprising an aliphatic alcohol may be used. For example, the oxidationstep may be performed in an aliphatic alcohol selected from the groupincluding, but not limited to, methanol, ethanol, n-propanol, andt-butanol. Alternatively, a solvent comprising an ether compound may beused. Suitable solvents comprising an ether compound may be selectedfrom the group including, but not limited to, ethyl ether,tetrahydrofuran, and dioxane. In another alternative, oxidation step maybe performed in a solvent comprising a hydrocarbon. Suitable solventscomprising a hydrocarbon may be selected from the group including, butnot limited to, pentane, hexane, benzene, and toluene. In anotheralternative, oxidation step may be performed in a solvent comprising ahalohydrocarbon. For example, dichloromethane may be used as a solvent.Alternatively, oxidation step may be performed in a solvent that is amixture of t-butanol and dichloromethane.

In another alternative, oxidation of the allylic carbon is performedusing CrO₂Cl₂ as an oxidizing agent and in the presence of3,5-dimethylpyrazole as a catalyst. Other oxidizing agents and catalystswithin the scope of the present invention will be known to one ofreasonable skill in the art. In another alternative, the acid-catalyzedisomerization step is performed with an acidic isomerization agent.Preferably, an acidic isomerization agent is selected from the groupincluding, but not limited to, mineral acids, organic protonic acids,and Lewis acids. Typically, the acidic isomerization agent is a mineralacid selected from the group including, but not limited to, phosphoricacid, sulfuric acid, perchloric acid, hydrohalide acids, andheteropolyacids. Preferably, the acidic isomerization agent is aheteropolyacid. Alternatively, the acidic isomerization agent isH₃[P(W₃O₁₀)₄]. In another alternative, the acidic isomerization agent isa hydrohalide acid selected from the group including, but not limitedto, hydrogen chloride, hydrogen bromide, and hydrogen iodide. In anotheralternative, the acidic isomerization agent is a carboxylic acid or asulfonic acid. Other examples of a suitable acidic isomerization agentinclude, but are not limited to, trifluoroacetic acid, acetic acid, andmethylsulfonic acid. A mixture of acetic acid and sulfuric acid may alsobe used as an acidic isomerization agent. Other acidic mixtures withinthe scope of the present invention will be known to one of reasonableskill in the art.

In a preferred embodiment, a solid phase acidic isomerization agent isused. For example, suitable solid phase acidic isomerization agentsinclude, but are not limited to, montmorillonite clay, acidic resins,Dowex® 50, Amberlyst® IR-15, and Nafion® perfluorinated resin.Additional examples of suitable acidic isomerization agents include, butare not limited to, acidic aluminum oxide, trifluoroboronetherate, tinchloride, and titanium tetrachloride.

An appropriate range of reaction time and reaction temperature isnecessary in order to produce a mixture of2-isopropyl-6,10-dimethyl-spiro[4.5]deca-2,6-dien-8-one (3) and2-isopropyl-6,10-dimethyl-spiro[4.5]deca-1,6-dien-8-one (4) frompremnaspirodiene (2) according to Scheme (1) below:

A reaction time that is too short will result in low yield of themixture of 2,6 diene and 1,6 diene isomers, since not all of thepremnaspirodiene will be converted to the desired end-product. Areaction time that is too long is uneconomical and presents problems forindustrial scale production of the desired end product. Typically, thereaction time preferred for production of the mixture of 2,6 diene and1,6 diene isomers is between about 1 hour and about 96 hours and thereaction temperature is between about 25 degrees Celsius and about 150degrees Celsius. More preferably, the reaction time is between about 2hours and about 24 hours. Most preferably, the reaction time is betweenabout 2 hours and about 6 hours. Appropriate reaction temperature issimilarly important. In the preferred embodiment, the reactiontemperature is between about 25 degrees Celsius and about 150 degreesCelsius. More preferably, the reaction temperature is between about 50degrees Celsius and about 125 degrees Celsius. Most preferably, thereaction temperature is between about 75 degrees Celsius and about 105degrees Celsius. In the preferred embodiment, the reaction time isbetween about 1 hour and about 96 hours and reaction temperature isbetween about 25 degrees Celsius and about 150 degrees Celsius.

In another embodiment the present invention describes a method forsynthesizing 2-isopropyl-6,10-dimethyl-spiro[4.5]deca-2,6-dien-8-one (3)and 2-isopropyl-6,10-dimethyl-spiro[4.5]deca-1,6-dien-8-one (4)comprising the steps of:

(1) subjecting (−)-premnaspirodiene (2) to oxidation of an allyliccarbon to form (−)-solavetivone (5); and

(2) subjecting the (−)-solavetivone (5) formed in step (1) toacid-catalyzed isomerization to form the mixture of2-isopropyl-6,10-dimethyl-spiro[4.5]deca-2,6-dien-8-one (3) and2-isopropyl-6,10-dimethyl-spiro[4.5]deca-1,6-dien-8-one (4).

In this alternative, the time and temperature conditions for step (2)are such that the predominant product is a mixture of2-isopropyl-6,10-dimethyl-spiro[4.5]deca-2,6-dien-8-one (3) and2-isopropyl-6,10-dimethyl-spiro[4.5]deca-1,6-dien-8-one (4). In thisalternative, the method can further comprise the step of separating2-isopropyl-6,10-dimethyl-spiro[4.5]deca-2,6-dien-8-one (3) and2-isopropyl-6,10-dimethyl-spiro[4.5]deca-1,6-dien-8-one (4) from themixture of 2-isopropyl-6,10-dimethyl-spiro[4.5]deca-2,6-dien-8-one (3)and 2-isopropyl-6,10[-dimethyl-spiro 4.5]deca-1,6-dien-8-one (4) inorder to produce the two positional isomers individually. Typically, theseparation step is performed by a method selected from the groupconsisting of thin-layer chromatography, column chromatography, gaschromatography, and countercurrent distribution. Other separation stepswithin the scope of the present invention will be known to one ofreasonable skill in the art.

In another embodiment the present invention describes a substantiallypurified and isolated mixture of2-isopropyl-6,10-dimethyl-spiro[4.5]deca-2,6-dien-8-one (3) and2-isopropyl-6,10-dimethyl-spiro[4.5]deca-1,6-dien-8-one (4). Thissubstantially purified and isolated mixture is prepared by the methoddescribed above and includes both positional isomers. As used hereinwith reference to this substantially purified and isolated mixture, theterm “substantially purified and isolated” means that the mixture issubstantially free of side products and unreacted components, althoughsolvents and other inert substances may remain. Although a specificdegree of purity is not intended by the use of the term “substantiallypurified and isolated,” typically, when solvents and other inertsubstances are removed from consideration of purity, the mixture formsat least about 75% of the compounds present. More typically, the mixtureforms at least about 85% of the compounds present. Preferably, themixture forms at least about 95% of the compounds present. Morepreferably, the mixture forms at least 97.5% of the compounds present.Still more preferably, the mixture forms at least 99% of the compoundspresent. Still more preferably, the mixture forms at least 99.5% of thecompounds present.

In another embodiment the present invention describes the substantiallypurified and isolated compound2-isopropyl-6,10-dimethyl-spiro[4.5]deca-2,6-dien-8-one represented byFIG. 3A, substantially free of2-isopropyl-6,10-dimethyl-spiro[4.5]deca-1,6-dien-8-one. As used hereinwith respect to this compound and with respect to the other positionalisomer 2-isopropyl-6,10-dimethyl-spiro[4.5]deca-1,6-dien-8-one, below,the term “substantially purified and isolated” means that thepreparation is substantially free of side products and unreactedcomponents, although solvents and other inert substances may remain.Although a specific degree of purity is not intended by the use of theterm “substantially purified and isolated,” typically, when solvents andother inert substances are removed from consideration of purity, thedesired compound (i.e.,2-isopropyl-6,10-dimethyl-spiro[4.5]deca-2,6-dien-8-one) forms at leastabout 75% of the compounds present. More typically, the desired compoundforms at least about 85% of the compounds present. Preferably, thedesired compound forms at least about 95% of the compounds present. Morepreferably, the desired compound forms at least 97.5% of the compoundspresent. Still more preferably, the desired compound forms at least 99%of the compounds present. Still more preferably, the desired compoundforms at least 99.5% of the compounds present.

In another embodiment the present invention describes the substantiallypurified and isolated compound2-isopropyl-6,10-dimethyl-spiro[4.5]deca-1,6-dien-8-one represented byFIG. 3B, substantially free of2-isopropyl-6,10-dimethyl-spiro[4.5]deca-2,6-dien-8-one. The term“substantially purified and isolated” with respect to this compound isas defined above with respect to the substantially purified and isolatedcompound 2-isopropyl-6,10-dimethyl-spiro[4.5]deca-2,6-dien-8-one.

In another embodiment the present invention describes a method forproducing (−)-premnaspirodiene (2) from a terpene substrate, comprisingthe steps of:

(1) providing the terpene substrate to a host cell transformed ortransfected with a vector comprising a sequence encoding a Hyoscyamusmuticus premnaspirodiene synthase gene;

(2) culturing the host cell under conditions suitable to produce(−)-premnaspirodiene from the terpene substrate; and

(3) isolating the (−)-premnaspirodiene.

Suitable production hosts may include, but are not limited to, anyorganism capable of expressing the genes required for thepremnaspirodiene production. Typically, the host cell will be amicroorganism cell or a plant cell. Other suitable host cells within thescope of the present invention will be known to one of reasonable skillin the art.

Microorganism host cells useful in the present invention for theproduction of premnaspirodiene may include, but are not limited to,bacteria, such as the enteric bacteria (Escherichia and Salmonella forexample) as well as Bacillus, Acinetobacter, Streptomyces,Methylobacter, Rhodococcus and Pseudomonas; Cyanobacteria, such asRhodobacter and Synechocystis; yeasts, such as Saccharomyces,Zygosaccharomyces, Kluyveromyces, Candida, Hansenula, Debaryomyces,Mucor, Pichia and Torulopsis; and filamentous fungi such as Aspergillusand Arthrobotrys, and algae for example. Preferably, the host cell is aeukaryotic cell. More preferably, the host cell is a yeast cell, whichis a eukaryotic microorganism host cell. Most preferably, the host cellis a Saccharomyces cerevisiae cell.

Microbial expression systems and expression vectors containingregulatory sequences that direct high level expression of foreignproteins are well known to those skilled in the art. These expressionsystems and expression vectors are known both for prokaryotic organismssuch as bacteria and for eukaryotic organisms such as yeast. Similarly,vectors or cassettes useful for the transformation of suitable microbialhost cells are well known in the art. These vectors and cassettes areknown both for prokaryotic organisms such as bacteria and for eukaryoticorganisms such as yeast. Typically, the vector or cassette containssequences directing expression of the relevant gene, a selectablemarker, and sequences allowing autonomous replication or chromosomalintegration. Suitable vectors comprise a region 5′ of the gene whichharbors transcriptional initiation controls and a region 3′ of the DNAfragment which controls transcriptional termination.

The Hyoscyamus muticus premnaspirodiene synthase gene is described in K.Back & J. Chappell, “Cloning and Bacterial Expression of a SesquiterpeneCyclase from Hyoscyamus muticus and Its Molecular Comparison to RelatedTerpene Cyclases,” J. Biol. Chem. 270: 7375-7381 (1995); K. Back & J.Chappell, “Identifying Functional Domains Within Terpene Cyclases Usinga Domain-Swapping Strategy, Proc. Natl. Acad. Sci. USA 93: 6841-6845(1996); and B. T. Greenhagen et al., “Identifying and ManipulatingStructural Determinates Linking Catalytic Specificities in TerpeneSynthases,” Proc. Natl. Acad. Sci. USA 103: 9826-9831 (2006), all ofwhich are incorporated by these references, and, as used herein,typically includes nucleic acid sequences encoding amino acid sequencesidentified in these references as part of active domains, includingcatalytic domains and domains responsible for selectivity of thesynthase reaction. As used herein, recitation of a nucleic acid sequenceencoding a specified amino acid sequence includes all possible codonsencoding that amino acid sequence in the absence of evidence suggestingthat any possible codon or combination of codons would be nonfunctionalin the step of either transcription of the DNA sequence or translationof the resulting mRNA.

Initiation control regions or promoters, which are useful to driveexpression of the relevant genes in the desired host cell are numerousand familiar to those skilled in the art. Termination control regionsmay also be derived from various genes native to the preferred hosts.

Expression of cloned heterologous genes in yeast cells, particularlycells of S. cerevisiae, is described in the following references, all ofwhich are incorporated herein by these references: S. D. Emr,“Heterologous Gene Expression in Yeast,” Meth. Enzymol. 185: 231-233(1991), is a general overview of expression in yeast, including thepossibility of exploiting protein secretion and modification in yeastand achieving stability of expressed proteins. A. B. Rose & J. R.Broach, “Propagation and Expression of Cloned Genes in Yeast: 2-μmCircle-Based Vectors, Meth. Enzymol. 185: 234-279 (1991), describes theuse of 2-μm circle-based vectors for transfection of genes into yeastand for expression of heterologous genes in yeast, including standard2-μm circle-based vectors, vectors for high copy propagation, vectorsfor expression of cloned genes in yeast, and vectors for specializedapplications. T. Stearns et al., “Manipulating Yeast Genome UsingPlasmid Vectors,” Meth. Enzymol. 185: 280-297 (1991), describes the useof yeast vector systems and components, the use of homologousrecombination to integrate plasmids into the yeast host genome, and theuse of centromere plasmids. L. M. Mylin et al., “Regulated GAL4Expression Cassette Providing Controllable and High-Level Output fromHigh-Copy Galactose Promoters in Yeast,” Meth. Enzymol. 185: 297-308(1991), describes the use of galactose-inducible promoters to providehigh levels of production of cloned proteins in yeast. V. L. Price etal., “Expression of Heterologous Proteins in Saccharomyces cerevisiaeUsing the ADH2 Promoter,” Meth. Enzymol. 185: 308-318 (1991), describesthe use of the glucose-repressible ADH2 promoter to providecontrollable, high level expression of cloned proteins in yeast. T.Etcheverry, “Induced Expression Using Yeast Copper MetallothioneinPromoter,” Meth. Enzymol. 185: 319-329 (1991), describes the use of theyeast CUP1 promoter to drive controllable expression of cloned genes inyeast. S. M. Kingsman et al., “High-Efficiency Yeast Expression VectorsBased on the Promoter of the Phosphoglycerate Kinase Gene,” Meth.Enzymol. 185: 329-341 (1991), describes the use of the yeast PGKpromoter to drive controllable expression of cloned genes in yeast. S.Rosenberg et al., “Glyceraldehyde-3-Phosphate Dehydrogenase-DerivedExpression Cassettes for Constitutive Synthesis of HeterologousProteins,” Meth. Enzymol. 185: 341-350 (1991), describes the use ofexpression cassette plasmids utilizing the strong GAPDH-491 promoter forhigh levels of heterologous protein production in yeast. A. Z.Sledziewski et al., “Superimposition of Temperature Regulation on YeastPromoters,” Meth. Enzymol. 185: 351-366 (1991), describes theconstruction of temperature-regulated variants of two strong yeastpromoters, TPI1 and ADH2, and the use of these promoters for regulationof expression and thus regulation of the extent of glycosylation ofproteins secreted by yeast. T. F. Donahue & A. M. Cigan, “Sequence andStructural Requirements for Efficient Translation in Yeast,” Meth.Enzymol. 185: 366-372 (1991), describes the significance of codon usagevariations between yeast and higher eukaryotes and the selection ofefficient leader sequences. E. W. Jones, “Vacuolar Proteases in YeastSaccharomyces cerevisiae,” Meth. Enzymol. 185: 372-386 (1991), describesthe elimination of vacuolar protease activity in yeast to maximize theyield of protein production from cloned genes. K. D. Wilkinson,“Detection and Inhibition of Ubiquitin-Dependent Proteolysis,” Meth.Enzymol. 185: 387-397 (1991), describes methods for preventingubiquitin-dependent protein degradation in yeast, again to maximize theyield of protein production from cloned genes. R. L. Kendall et al.,“Cotranslational Amino-Terminal Processing,” Meth. Enzymol. 185: 398-407(1991), describes the cotranslational processing events that occur inyeast at the amino-termini of nascent polypeptide genes and theireffects on heterologous gene expression and protein stability. A. J.Brake, “α-Factor Leader-Directed Secretion of Heterologous Proteins fromYeast,” Meth. Enzymol. 185: 408-421 (1991), describes expression systemsbased on the yeast α-factor leader. R. A. Hitzeman et al., “Use ofHeterologous and Homologous Signal Sequences for Secretion ofHeterologous Proteins from Yeast,” Meth. Enzymol. 185: 421-440 (1991),describes the use of both heterologous and homologous signal sequencesfor the production and secretion of heterologous gene products in yeast.V. Chisholm et al., “Molecular and Genetic Approach to Enhancing ProteinSecretion,” Meth. Enzymol. 185: 471-482 (1991), describes the use of anenhanced secretion phenotype occurring among drug-resistant yeastmutants to maximize secretion of cloned proteins in yeast.

General molecular biological techniques of gene cloning, site-directedmutagenesis, and fusion protein construction can be used to providenucleic acid segments that include therein the Hyoscyamus muticuspremnaspirodiene synthase gene. Typically, the nucleic acid segments areDNA nucleic acid segments. Typically, as described above, the Hyoscyamusmuticus premnaspirodiene synthase gene is operatively linked to at leastone nucleic acid expression control element, such as, but not limitedto, a promoter, an enhancer, or a site capable of binding a repressor oractivator. Such nucleic acid expression control elements are well knownin the art. Typically, as described above, the Hyoscyamus muticuspremnaspirodiene synthase gene is included in a vector and, as such, isagain operatively linked to at least one nucleic acid expression controlelement. Site-directed mutagenesis can be used, for example, to provideoptimum codon selection for expression in S. cerevisiae, as describedabove. The Hyoscyamus muticus premnaspirodiene synthase gene can, in onealternative, be expressed in the form of a nucleic acid segment encodinga fusion protein, such as a purification tag or other detectable proteindomain.

Where commercial production of premnaspirodiene is desired, a variety offermentation methodologies may be applied. For example, large scaleproduction may be effected by either batch or continuous fermentation. Aclassical batch fermentation is a closed system where the composition ofthe media is set at the beginning of the fermentation and not subject toartificial alterations during the fermentation. Thus, at the beginningof the fermentation the medium is inoculated with the desiredmicroorganism or microorganisms and fermentation is permitted to occurwithout further addition of nutrients. Typically, the concentration ofthe carbon source in a batch fermentation is limited, and factors suchas pH and oxygen concentration are controlled. In batch systems themetabolite and biomass compositions of the system change constantly upto the time the fermentation is stopped. Within batch cultures cellstypically modulate through a static lag phase to a high growth log phaseand finally to a stationary phase where growth rate is diminished orhalted. If untreated, cells in the stationary phase will eventually die.

A variation on the standard batch system is the Fed-Batch system.Fed-Batch fermentation processes are also suitable for use in thepresent invention and comprise a typical batch system with the exceptionthat nutrients are added as the fermentation progresses. Fed-Batchsystems are useful when catabolite repression is apt to inhibit themetabolism of the cells and where it is desirable to have limitedamounts of substrate in the medium. Also, the ability to feed nutrientswill often result in higher cell densities in Fed-Batch fermentationprocesses compared to Batch fermentation processes. Factors such as pH,dissolved oxygen, nutrient concentrations, and the partial pressure ofwaste gases such as CO₂ are generally measured and controlled inFed-Batch fermentations. Batch and Fed-Batch fermentations are commonand well known in the art and examples may be found in Brock, T. D.;Biotechnology: A Textbook of Industrial Microbiology, 2nd ed.; SinauerAssociates: Sunderland, Mass., 1989; or Deshpande, M. V. Appl. Biochem.Biotechnol. 36:227 (1992), herein incorporated by reference.

Commercial production of premnaspirodiene may also be accomplished withcontinuous fermentation. Continuous fermentation is an open system wherea defined fermentation medium is added continuously to a bioreactor andan equal amount of conditioned medium is removed simultaneously forprocessing. Continuous fermentation generally maintains the cultures ata constant high density where cells are primarily in their log phase ofgrowth. Continuous fermentation allows for modulation of any number offactors that affect cell growth or end product concentration. Forexample, one method will maintain a limiting nutrient such as the carbonsource or nitrogen level at a fixed rate and allow all other parametersto moderate. In other systems a number of factors affecting growth canbe altered continuously while the cell concentration, measured by themedium turbidity, is kept constant. Continuous systems strive tomaintain steady state growth conditions and thus the cell loss due tothe medium removal must be balanced against the cell growth rate in thefermentation. Methods of modulating nutrients and growth factors forcontinuous fermentation processes as well as techniques for maximizingthe rate of product formation are well known in the art of industrialmicrobiology and a variety of methods are detailed by Brock, supra.

In the preceding embodiments, typically the terpene substrate isfarnesyl diphosphate. Preferably, the host cell is a yeast cell thatoverproduces farnesyl diphosphate. Most preferably, the host cell is aSaccharomyces cerevisiae cell that overproduces farnesyl diphosphate.Other suitable host cells and substrates within the scope of the presentinvention will be known to one of reasonable skill in the art.

Typically, the step of isolating the premnaspirodiene produced by thehost cell is performed by: (i) sequestering the premnaspirodiene bybinding it to a hydrophobic resin; (ii) and isolating thepremnaspirodiene from the hydrophobic resin. Preferably, the hydrophobicresin is Amberlite® XAD-16 hydrophobic resin. Other hydrophobic resinswithin the scope of the present invention will be known to one ofreasonable skill in the art.

Typically, premnaspirodiene is isolated from the hydrophobic resin bymethanol extraction. Other methods of isolating premnaspirodiene thatare within the scope of the present invention will be known to one ofreasonable skill in the art.

In an alternative, a two-phase system can be used with a non-polarsolvent, substantially immiscible with an aqueous phase, added to thefermentation broth and the premnaspirodiene removed from the non-polarphase by distillation. A preferred non-polar solvent is an oil. Aparticularly preferred oil is a vegetable oil such as soybean oil.Alternative non-polar solvents include, but are not limited to, highmolecular weight aliphatic hydrocarbons such as, but not limited to,dodecane, tridecane, tetradecane, pentadecane, and hexadecane; eitherstraight-chain or branched-chain isomers can be used; thesehigh-molecular weight aliphatic hydrocarbons are optionally substitutedwith one or more hydroxy or halogen substituents as long as thesubstituted hydrocarbon remains substantially immiscible with theaqueous phase.

Others have reported the use of two-phase systems in bacterial wholecell transformations (R. J. Sowden et al., “Biotransformation of theSesquiterpene (+)-Valencene by Cytochrome P450_(cam) and P450_(BM-3) ,”Org. Biomol. Chem. 3: 57-64 (2005)) and in yeast fermentations (A.-L.Lindahl et al., “Production of Artemisinin Precursor Amorpha-1,4-dieneby Engineered Saccharomyces cerevisiae,” Biotechnol. Lett. 28: 571-580(2006)). In those cases the hydrocarbons hexadecane and dodecane wereused.

In another embodiment the present invention describes a method forsynthesizing 5-epi-β-vetivone (1) comprising the steps of:

(1) providing a terpene substrate to a host cell transformed ortransfected with a vector comprising a sequence encoding a Hyoscyamusmuticus premnaspirodiene synthase gene;

(2) culturing the host cell under conditions suitable to produce(−)-premnaspirodiene from the terpene substrate;

(3) isolating the (−)-premnaspirodiene (2);

(4) subjecting the isolated (−)-premnaspirodiene (2) to oxidation of anallylic carbon to form (−)-solavetivone (5); and

(5) subjecting the (−)-solavetivone (5) formed in step (4) toacid-catalyzed isomerization to form 5-epi-β-vetivone (1);

wherein time and temperature conditions for step (5) are such that thepredominant product is 5-epi-β-vetivone (1).

As used herein, the term “providing a terpene substrate” can include:(1) provision of an extraneous terpene substrate to the host cells; or(2) having the host cells themselves synthesize the terpene substrate sothat the terpene substrate can be converted into the(−)-premnaspirodiene (2). Typically, the host cells synthesize theterpene substrate.

In another embodiment the present invention describes a method forsynthesizing a mixture of2-isopropyl-6,10-dimethyl-spiro[4.5]deca-2,6-dien-8-one (3) and2-isopropyl-6,10[-dimethyl-spiro 4.5]deca-1,6-dien-8-one (4) comprisingthe steps of

(1) providing a terpene substrate to a host cell transformed ortransfected with a vector comprising a sequence encoding a Hyoscyamusmuticus premnaspirodiene synthase gene as described above;

(2) culturing the host cell under conditions suitable to produce(−)-premnaspirodiene from the terpene substrate;

(3) isolating the (−)-premnaspirodiene (2);

(4) subjecting the isolated (−)-premnaspirodiene (2) to oxidation of anallylic carbon to form (−)-solavetivone (5); and

(5) subjecting the (−)-solavetivone (5) formed in step (4) toacid-catalyzed isomerization to form the mixture of2-isopropyl-6,10-dimethyl-spiro[4.5]deca-2,6-dien-8-one (3) and2-isopropyl-6,10-dimethyl-spiro[4.5]deca-1,6-dien-8-one (4);

wherein time and temperature conditions for step (5) are such that thepredominant product is the mixture of2-isopropyl-6,10-dimethyl-spiro[4.5]deca-2,6-dien-8-one (3) and2-isopropyl-6,10-dimethyl-spiro[4.5]deca-1,6-dien-8-one (4).

Similar methods, with an additional step of isolating the positionalisomers, can be used to synthesize2-isopropyl-6,10-dimethyl-spiro[4.5]deca-2,6-dien-8-one or2-isopropyl-6,10-dimethyl-spiro[4.5]deca-1,6-dien-8-one.

In general, the method for synthesizing2-isopropyl-6,10-dimethyl-spiro[4.5]deca-2,6-dien-8-one comprises thesteps of:

(1) providing a terpene substrate to a host cell transformed ortransfected with a vector comprising a sequence encoding a Hyoscyamusmuticus premnaspirodiene synthase gene;

(2) culturing the host cell under conditions suitable to produce(−)-premnaspirodiene from the terpene substrate; and

(3) isolating the (−)-premnaspirodiene;

(4) subjecting the isolated (−)-premnaspirodiene to oxidation of anallylic carbon to form (−)-solavetivone; and

(5) subjecting the (−)-solavetivone formed in step (4) to acid-catalyzedisomerization to form a mixture of2-isopropyl-6,10-dimethyl-spiro[4.5]deca-2,6-dien-8-one and2-isopropyl-6,10-dimethyl-spiro[4.5]deca-1,6-dien-8-one; wherein timeand temperature conditions for step (5) are such that the predominantproduct is the mixture of 2-isopropyl-6,10[-dimethyl-spiro4.5]deca-2,6-dien-8-one and2-isopropyl-6,10-dimethyl-spiro[4.5]deca-1,6-dien-8-one; and

(6) isolating the2-isopropyl-6,10-dimethyl-spiro[4.5]deca-2,6-dien-8-one from the mixtureof 2-isopropyl-6,10-dimethyl-spiro[4.5]deca-2,6-dien-8-one and2-isopropyl-6,10-dimethyl-spiro[4.5]deca-1,6-dien-8-one formed in step(5) by a method selected from the group consisting of thin-layerchromatography, column chromatography, gas chromatography, andcountercurrent distribution.

In general, the method for synthesizing2-isopropyl-6,10-dimethyl-spiro[4.5]deca-1,6-dien-8-one comprises thesteps of:

(1) providing a terpene substrate to a host cell transformed ortransfected with a vector comprising a sequence encoding a Hyoscyamusmuticus premnaspirodiene synthase gene;

(2) culturing the host cell under conditions suitable to produce(−)-premnaspirodiene from the terpene substrate; and

(3) isolating the (−)-premnaspirodiene;

(4) subjecting the isolated (−)-premnaspirodiene to oxidation of anallylic carbon to form (−)-solavetivone; and

(5) subjecting the (−)-solavetivone formed in step (4) to acid-catalyzedisomerization to form a mixture of2-isopropyl-6,10-dimethyl-spiro[4.5]deca-2,6-dien-8-one and2-isopropyl-6,10-dimethyl-spiro[4.5]deca-1,6-dien-8-one; wherein timeand temperature conditions for step (5) are such that the predominantproduct is the mixture of2-isopropyl-6,10-dimethyl-spiro[4.5]deca-2,6-dien-8-one and2-isopropyl-6,10-dimethyl-spiro[4.5]deca-1,6-dien-8-one; and

(6) isolating the2-isopropyl-6,10-dimethyl-spiro[4.5]deca-1,6-dien-8-one from the mixtureof 2-isopropyl-6,10-dimethyl-spiro[4.5]deca-2,6-dien-8-one and2-isopropyl-6,10-dimethyl-spiro[4.5]deca-1,6-dien-8-one formed in step(5) by a method selected from the group consisting of thin-layerchromatography, column chromatography, gas chromatography, andcountercurrent distribution.

The present invention also includes a fragrance composition with acompound having the structure:

in an amount effective to impart a fragrance. In one aspect, thecompound of the fragrance composition is present in an amount of atleast 0.01% by weight. In another aspect, the fragrance compositioncomprises an amount of the compound of Formula (1) effective to impartfragrance in combination with conventional fragrance ingredients.

Another object of the present invention is a perfumed product comprisinga compound having the structure of Formula (1). The perfumed product canbe a household product, such as, for example, a solid or liquiddetergent, a fabric softener, an air freshener, a fabric refresher, anironing water, a paper, a wipe or a bleach. The perfumed product can bea cosmetic product or a body care product, for example a perfume, acologne or after-shave lotion, a perfumed soap, a shower or bath salt,mousse, oil or gel, a hygiene product, a hair care product, a shampoo, adeodorant or antiperspirant.

The present invention can be a perfuming composition containing thecompound of Formula (1) in an amount sufficient to give a fragrance tothe composition. In one embodiment, the perfuming composition canadditionally contain at least one perfumery adjuvant.

Another aspect of the present invention is a fragrance applicationcomprising a compound of Formula (1). The fragrance application can be,for example, a household product such as laundry product, a solid orliquid detergent, a fabric softener, an air freshener, a fabricrefresher, an ironing water, a paper, a wipe or a bleach. In anotheraspect, the fragrance application can be a cosmetic product or body careproduct such as a perfume, a cologne or after-shave lotion, a perfumedsoap, a shower or bath salt, mousse, oil or gel, a hygiene product, ahair care product, a shampoo, a deodorant or antiperspirant.

The invention also includes methods of imparting a woody, grapefruit, orvetivent odor to a fragrance or fragrance composition by providing thecompound of Formula (1) to the fragrance.

Methods of the invention include imparting, improving, enhancing ormodifying a fragrance formulation through the addition of an olfactoryacceptable amount of Formula (1). The olfactory acceptable amount can befrom about 0.005 to about 10 weight percent of the fragranceformulation. In one aspect, the olfactory acceptable amount is fromabout 0.5 to about 8 weight percent of the fragrance formulation. Inanother aspect, the method of claim 18, wherein the olfactory acceptableamount is from about 1 to about 7 weight percent of the fragranceformulation.

As mentioned above, the invention provides for the synthesis ofcompounds to be used as a perfuming ingredient. In another embodiment,the invention describes a method to confer, enhance, improve or modifythe odor properties of a perfuming composition or of a perfumed article.The method comprises adding an effective amount of the fragrantcomponent described by the formulas to said perfumed compositions orarticle.

The compounds, which are perfuming compositions that can beadvantageously employed as perfuming ingredients, are also an object ofthe present invention. The fragrance or perfume composition can alsocontain additional ingredients such as a perfumery carrier, a perfumerybase; or one or more one perfumery adjuvants. Examples of fragrance andperfuming compositions can be found in, for example, Arctander, S.,Perfume and Flavor Chemicals (Montclair, N.J., 1969), Arctander, S.,Perfume and Flavor Materials of Natural Origin (Elizabeth, N.J., 1960)and in “Flavor and Fragrance Materials—1991,” Allured Publishing Co.Wheaton, 111. USA, which are herein incorporated by reference.

The terms “fragrance” or “perfume” means a discernible odor at normalroom temperature (about 25° C.) that is generally regarded asinteresting, pleasant or attractive. The term “perfumery carrier” meansa material which is practically neutral from a perfumery point of view,i.e. that does not significantly alter the organoleptic properties ofperfuming ingredients. The carrier may be a liquid or a solid.

A liquid carrier may be, for example, an emulsifying system, i.e. asolvent and a surfactant system, or a solvent commonly used inperfumery. A solvent can be, for example, a dipropyleneglycol, diethylphthalate, isopropyl myristate, benzyl benzoate,2-(2-ethoxyethoxy)-1-ethanol, ethyl citrate, water/ethanol mixtures,limonene or other terpenes, isoparaffins, glycol ethers and glycol etheresters.

A solid carrier may be, for example, absorbing gums or polymers, or yetencapsulating materials, such as wall-forming and plasticizingmaterials, such as mono, di- or trisaccharides, natural or modifiedstarches, hydrocolloids, cellulose derivatives, polyvinyl acetates,polyvinylalcohols, proteins or pectins, or the like. Encapsulation is aprocess which is well known to a person skilled in the art, and may beperformed, for example, using techniques such as spray-drying,agglomeration or yet extrusion; or consists of a coating encapsulation,including coacervation and complex coacervation techniques.

The term “perfumery base” means compositions comprising at least oneperfuming co-ingredient. A “perfuming co-ingredient” can be a compoundused in a perfuming preparation or composition known by one of skill inthe art to impart or modify the odor of a composition. The compounds ofthe invention exhibit interesting fragrance properties or odorcharacteristics, and may be used to impart, improve, enhance or modifythe odor of a wide variety of products, or it may be used as a componentof a perfume (or fragrance composition) to contribute its odor characterto the overall odor of such perfume. For the purposes of this inventiona perfume can be a mixture of fragrance materials which is used toimpart a desired odor to the skin and/or any product for which anagreeable odor is desirable.

It is contemplated that more than one perfume compound or compositioncan be used in the methods or articles of the present invention. Thenature and type of the perfuming co-ingredients can be selected by oneof skill in the art according to the intended use or application and theorganoleptic effect desired. In general terms, these perfumingco-ingredients belong to chemical classes as varied as alcohols,aldehydes, ketones, esters, ethers, acetates, nitrites, terpenehydrocarbons, nitrogenous or sulphurous heterocyclic compounds andessential oils, and said perfuming co-ingredients can be of natural orsynthetic origin. It is also understood that said co-ingredients mayalso be compounds known to release in a controlled manner various typesof perfuming compounds.

Examples of perfumed articles include, but are not limited to, solid orliquid detergents, a fabric softener, an air freshener, a fabricrefresher, ironing water, a paper, a wipe or a bleach The perfumedarticles can be intended for domestic or industrial use. Other perfumedarticles can be, for example, perfumes, colognes or after-shave lotions,perfumed soaps, shower or bath salts, mousses, oils or gels, hygieneproducts or hair care products, deodorants or antiperspirants, andcosmetic preparations.

The invention also includes a mixture comprising the compound of Formula(1) and an auxiliary ingredient compatible with the compound of Formula(1), the weight ratio of the compound of Formula (1) being in the rangeof from about 1:1 up to about 1:5. As used herein, an “auxiliaryingredient” can be, for example, thickeners, vitamins, provitamins,anti-grease agents, antioxidants, preservatives, perfumes and UV-lightabsorbing inorganic pigments. As used herein, “weight ratio” refers to amixture amount.

The invention also includes a fragrance modifying composition comprisingthe compound of Formula (1) and an auxiliary ingredient compatible withthe compound of Formula (1), the weight ratio of the compound of Formula(1) being in the range of from about 1:1 up to about 1:5.

Also included in the invention is a perfume composition comprising thecompound of Formula (1) and at least one compatible adjuvant, the ratioof the compound of Formula (1) being in the range of from about 1:1 upto about 1:5. As used herein, a “compatible adjuvant” can be, forexample, thickeners, vitamins, provitamins, anti-grease agents,antioxidants, preservatives, perfumes and UV-light absorbing inorganicpigments.

The invention also includes a cologne composition comprising thecompound of Formula (1) and at least one compatible adjuvant, the ratioof the compound of Formula (1) being in the range of from about 1:1 upto about 1:5. Additionally, the invention includes a method of using thecompound of Formula (1) as a perfume. In one embodiment, the method ofusing the compound of Formula (1) as a perfume further comprises anauxiliary ingredient compatible with the compound of Formula (1).

The following examples are provided for the purpose of illustrating thepresent invention and are not to be considered limiting.

Example 1 Production of Premnaspirodiene (2)

In this example premnaspirodiene was produced by expressing theHyoscyamus muticus premnaspirodiene synthase (the “HPS”) gene inSaccharomyces cerevisiae.

The HPS gene was cloned into the yeast shuttle expression vectorYEp-GW-URA-NheI/BamHI as described in (1) to give YEp-HPS-ura. Thisvector contained the ADH1 promoter for initiating transcription of theHPS gene. In addition, it contained the ADH1 terminator downstream ofthe HPS gene. This vector was maintained in S. cerevisiae by selectingmedia lacking uracil and it was maintained in E. coli by selecting forresistance to ampicillin.

For production, YEp-HPS-ura was transformed into Cali-5 using the yeasttransformation kit (Sigma). Transformants were selected on SDE-uramedium (0.67% Bacto yeast nitrogen base without amino acids, 2% glucose,0.14% yeast synthetic drop-out medium supplement without uracil, 40 mg/Lergosterol). Colonies were picked and screened for premnaspirodieneproduction.

Cali-5 was previously engineered to overproduce farnesyl diphosphate(the “FPP”), the substrate for the HPS enzyme. It contained thefollowing mutations: erg9, sue, dppl. It also contained approximately 8copies of the truncated HMG2 gene. The truncated form of HMG2 is drivenby the GPD promoter and no longer under tight regulation and allows foran increase in carbon flow to FPP. Cali-5 is a derivative of SW23B,described in U.S. Pat. No. 6,689,593 to Millis et al., incorporated bythis reference.

Due to the volatile nature of sesquiterpenes, it is necessary tosequester them during the fermentation process. One such sequestrationmethod is the addition of the hydrophobic resin Amberlite XAD-16(Sigma). This resin has been used in fermentation of othermicroorganisms to capture natural products as they are produced (2, 3,4).

Production was carried out in a 14 L fermentation tank (Bioflow 110).Eight liters of fermentation medium was prepared and autoclaved in thefermentation tank (160 g (NH₄)₂SO₄, 160 g KH₂PO₄, 8 g NaCl, 48 gMgSO₄.7H₂O, 32 g yeast extract (Difco). Afterwards the followingcomponents were added: 160 ml mineral solution (FeSO₄.7H₂O 0.028%,ZnSO₄.7H₂O 0.029%, CuSO₄.5H₂O 0.008%, Na₂MoO₄.2H₂O 0.024%, CoCl₂.6H₂O0.024%, MnSO₄H₂O 0.017%, HCl 1 ml), 80 ml 50% glucose, 240 ml vitaminsolution (biotin 0.001%, Ca-pantothenate 0.012%, inositol 0.06%,pyridoxine-HCl 0.012%, thiamine-HCl 0.012%), 80 ml 10% CaCl₂, and 200 gautoclaved XAD-16 beads; the beads were autoclaved in water and thewater removed before adding them to the medium. Finally, 8 ml of 50mg/ml ergosterol in 100% ethanol was added. The ergosterol was mixedwith the 100% ethanol and incubated in a hot water bath to aid in thedissolving of the ergosterol. Although the ergosterol does notcompletely dissolve in the ethanol, the complete mixture was added tothe medium.

The seed culture for inoculating the fermentation medium was prepared byinoculating 50 ml of SDE-ura medium with Cali-5 YEp-HPS-ura. Thisculture was grown until early stationary phase (24-48 hr). One ml ofthis culture was inoculated into 500 ml of SDE-ura medium and grown for24 hr. A 400 ml aliquot (5% inoculum) was used to inoculate the 8 L ofmedium.

The fermentor was maintained at 28° C. The air flow was 4.5 L/min andthe pO₂ was maintained above 20% by adjusting the rpm. Furthermore, pHwas maintained at 4.5 using acetic acid and NaOH.

Once the glucose was below 1 g/L, after approximately 14 hours, aglucose feed was attached and fed at a rate of 30 ml/hr. The glucosefeed was made by mixing 1400 ml 60% glucose and 328 ml 12.5% yeastextract.

After 5 days, the air and agitation were turned off, and the XAD-16resin was allowed to settle to the bottom of the tank, for approximately30 minutes. The broth was then siphoned off and the beads collected in a2 L flask. The beads were washed with 2×2 L of deionized water. Afterremoving as much of the water with a pipette as was possible, 250 ml of100% methanol was added. The mixture was incubated at room temperaturefor at least an hour and then the methanol extract was removed anddiscarded. To elute the premnaspirodiene, 4×500 ml of methanol was used.After the addition of methanol for each extraction, the beads andmethanol were warmed to 45° C. to aid in the recovery. The methanolextracts were pooled and concentrated in vacuo to 500 to 800 ml.

Because a significant amount of premnaspirodiene remained attached tothe cells, the medium from the fermentation tank was allowed to sit inlarge glass vessels for 1 to 2 days at room temperature. This was enoughtime for the yeast cells to settle to the bottom of the tank. At thispoint, most of the medium was siphoned off. The remaining broth andcells were then collected by centrifugation.

To the cell pellet was added 250 ml of acetone. The cells wereresuspended and the mixture was mixed for 10 minutes. The mixture wascentrifuged for 10 minutes to pellet the cells. The acetone was removedand another 250 ml of acetone was added to the cell pellet. The solutionwas mixed for 10 minutes and centrifuged again. The acetone was removedand added to the first acetone extract. The acetone pools wereconcentrated in vacuo to approximately 250 ml.

The acetone extract was mixed with the methanol extract. Thispremnaspirodiene was extracted from the acetone/methanol solution with3×300 ml of pentane. The pentane extracts were pooled and concentratedin vacuo to afford a solution of premnaspirodiene 40-50% pure. The crudematerial was purified on a silica gel column (100% hexane) to afford thetitle compound as a colorless oil (3.35 g, 91.4% pure by GC/MS). ¹H NMR(CDCl₃): 0.91 (d, 3H), 1.43 (m, 1H), 1.57 (m, 4H), 1.68 (s, 3H), 1.73(m, 6H), 1.84 (m, 2H), 2.01 (m, 1H), 2.47 (m, 1H), 4.71 (d, 2H), 5.29(s, 1H); ESIMS m/z 205 (M+H).

Example 2 Production of Premnaspirodiene Using Vegetable Oil asSequestering Agent

As described in Example 1, YEp-HPS-ura was transformed into strainsCALI-5 or ALX7-95, a leucine prototrophic derivative of CALI-5, usingthe yeast transformation kit (Sigma). Transformants were selected onSDE-ura medium (0.67 Bacto yeast nitrogen base without amino acids, 2%glucose, 0.14% yeast synthetic drop-out medium without uracil, 40 mg/Lergosterol). Colonies were picked and screened for premnaspirodieneproduction.

Due to the volatile nature of sesquiterpenes, it is necessary tosequester them during the fermentation process. A second suchsequestration method is the addition of vegetable oil to the fermentorto capture the nonpolar sesquiterepene hydrocarbon.

Production was carried out in a 14-L fermentation tank (Bioflow 110).Eight liters of fermentation medium was prepared and autoclaved in thefermentation tank (160 g (NH₄)₂SO₄, 160 g KH₂PO₄, 8 g NaCl, MgSO₄.7H₂O,32 g yeast extract (Difco). Afterward the following components wereadded: 160 ml mineral solution (0.028% FeSO₄.7H₂O, 0.029% ZnSO₄.7H₂O,0.008% CuSO₄.5H₂O, 0.024% Na₂MoO₄.2H₂O, 0.024% CoCl₂.6H₂O, 0.017%MnSO₄H₂O, 1 mL HCl); 80 mL 50% glucose; 240 mL vitamin solution (0.001%biotin; 0.012% calcium pantothenate, 0.06% inositol, 0.012%pyridoxine-HCl, 0.012% thiamine-HCl); 80 mL 10% CaCl₂, and 200 mLautoclaved soybean oil (purchased from local groceries). Finally, 8 mLof 50 mg/mL cholesterol in 100% ethanol was added.

The seed culture for inoculating the fermentation medium was prepared byinoculating 50 mL of SDE-ura medium with CALI-5 or ALX7-95 containingYEp-HPS-ura. This culture was grown until early stationary phase (24-48hr). One mL of this culture was inoculated into 500 mL of SDE-ura mediumand grown for 24 hr. A 400-mL aliquot (5% inoculum) was used toinoculate the 8 L of medium.

The fermentor was maintained at 26° C. The air flow was 4.5 L/min andthe dO₂ was maintained above 30% by adjusting the rpm. Furthermore, thepH was maintained at 4.5 using acetic acid and NaOH.

Once the glucose concentration was below 1 g/L, a feeding regimen wasinitiated such that the glucose in the fermentor was kept between 0 and1 g/L. The glucose feed was made by mixing 1400 mL of 60% glucose and328 mL of 12.5% yeast extract.

After 5 days, the air and agitation were turned off, and the oil wasallowed to rise to the top of the tank and decanted.

Example 3 Preparation of2-Isopropenyl-6,10-dimethyl-spiro[4.5]dec-6-en-8-one (the“(−)-solavetivone”) (5)

3,5-Dimethylpyrazole (47 g, 0.49 mol) was dissolved in a mixture ofCH₂Cl₂ (650 mL) and t-butyl alcohol (31 mL). The solution was thencooled to ±78° C. Chromyl chloride (CrO₂Cl₂) (13.3 mL) was added over 15min and stirred for another 15 min before it was allowed to warm to roomtemperature. Premnaspirodiene (6.69 g, 32.7 mmol) was dissolved inCH₂Cl₂ (650 mL) and added rapidly to the reaction. The dark red solutionwas stirred for 48 hours. The reaction was concentrated under vacuum andthe residue was suspended in ether. The suspension was then filteredthrough Celite to remove most of the chromium. The supernatant was thendiluted to a 1:1 mixture with hexane. The suspension was filteredthrough a pad of silica to remove some of the 3,5-dimethylpyrazole.After evaporation under vacuum, the residue was purified on a silica gelcolumn (hexane:ether, 9:1, hexane:ether, 2:1) to afford the titlecompound as a light yellow oil (3.45 g, 48.3%). ¹H NMR (CDCl₃): 1.00 (d,3H), 1.59 (m, 2H), 1.68 (m, 1H), 1.76 (s, 3H), 1.93 (m, 5H), 2.11 (m,2H), 2.21 (dd, 1H), 2.55 (m, 1H), 2.66 (dd, 1H), 4.74 (dd, 2H), 5.75 (s,1H); ESIMS m/z 219 (M+H).

Example 4 Preparation of2-Isopropylidene-6,10-dimethyl-spiro[4.5]dec-6-en-8-one (the“5-epi-β-vetivone”) (1)

To a solution of (−)-solavetivone (5) (1.66 g, 7.62 mmol) dissolved inethanol (16 mL) was added Amberlyst® IR-15 (1.66 g). The suspension wasthen heated at 100° C. in a sealed reaction flask for 50 min. Thesuspension was then filtered through Celite and evaporated under vacuum.The residue was purified on a silica gel column (hexane:ether, 85:15).Fractions were analyzed by GC/MS and only fractions with a purityof >95% were retained. Fractions with purities of 50-94% were combinedand repurified. This process was repeated until 3 silica gel columnshave been run. Final consolidation of all three purifications yieldedthe title compound as a colorless oil (700 mg, 42.2%). ¹H NMR (CDCl₃):0.93 (d, 3H), 1.58 (s, 6H), 1.82 (m, 3H), 1.86 (s, 3H), 2.05 (m, 1H),2.16 (dd, 1H), 2.37 (m, 3H), 2.57 (dd, 1H), 5.75 (s, 1H); ESIMS m/z 219(M+H).

Example 5 Preparation of2-Isopropyl-6,10-dimethyl-spiro[4.5]deca-2,6-dien-8-one (3) &2-Isopropyl-6,10-dimethyl-spiro[4.5]deca-1,6-dien-8-one (4).

To a solution of (−)-solavetivone (5) (100 mg, 0.46 mmol) dissolved inethanol (2 mL) was added Amberlyst® IR-15 (150 mg). The suspension wasthen heated at 105° C. in a sealed reaction flask for 96 hours. Thesuspension was then filtered through Celite and evaporated under vacuum.The residue was purified on a silica gel column (hexane:ether, 85:15) toafford the mixture as a colorless oil (67 mg, 67%). ESIMS m/z 219 (M+H),78.7% at 14.71 min; 219 (M+H), 17.1% at 14.89 min.

Example 6 Oxidation of (+)-Valencene to (+)-Nootkatone

In order to test various reaction conditions for the oxidation ofpremnaspirodiene to solavetivone, reactions were carried out oncommercially available valencene, a compound that is chemically similarto premnaspirodiene and would be expected to oxidized under similarreaction conditions. Reactions were carried out using 250 mg of startingmaterial in a single reaction, using combinations of sodium chlorite andeither t-butylhydroperoxide (t-BuOOH) or N-hydroxyphthalimide (NHPI) asdescribed (S. M. Silvestre & J. A. R. Salvador, “Allylic and BenzylicOxidation Reactions with Sodium Chlorite,” Tetrahedron 63: 2439-2445(2007)). The conditions used were those shown in Table 1. Of theconditions tested, that used in Experiment 1a (Table 1) gave the highestyield and the fewest byproducts. In Experiment 1a, the only byproductobserved was unreacted starting material.

TABLE 1 Oxidation of (+)-Valencene to (+)-Nootkatone NaClO₂ t-BuOOH NHPI% Product # of Exp (equiv) (equiv) (equiv) hplc byproducts 1a 1.2 5.0 NA75% 1 1b 1.2 3.0 NA 65% 1 1c 2.1 5.0 NA 45% 2 1d 2.5 5.0 NA 45% 2 2a 1.5NA 0.1 60% 3 2b 1.5 NA  0.5. 65% 3 2c 2.0 NA 0.1 40% 3 2d 3.0 NA 0.1 25%3

In a first oxidation procedure on a larger quantity of (+)-valencenecarried out according to Experiment 1a of Table 1, (+)-valencene (1.0 g,4.9 mmol, 1.0 equiv.) was placed in a four-neck 250-mL flask equippedwith a nitrogen inlet tube, thermowell, and a mechanical stirrer.Acetonitrile (45 mL) and water (15 mL) were added to the flask. A 70%solution in water of t-butyl hydroperoxide (3.15 g, 24.5 mmol, 5 equiv.)was added to the solution followed by the slow addition of sodiumchlorite (80%) (0.66 g, 5.9 mmol, 1.2 equiv.). The reaction was heatedto 50° C. for 18 h; it was then cooled and poured info a 10% solution ofsodium sulfite (100 mL). This solution was then extracted with diethylether (100 mL). The organic layer was washed with saturated sodiumbicarbonate (50 mL), dried over sodium sulfate and absorbed onto silicagel (2 g). This material was then purified on a silica gel column (20 g)and eluted with 5% ethyl acetate in heptane (250 mL) and 10% ethylacetate in heptane (250 mL). The fractions containing the desiredproduct were combined and concentrated under reduced pressure to givethe desired product in 72% yield (0.48 g).

In a second oxidation procedure on a larger quantity of (+)-valencenecarried out substantially according to Experiment 2a of Table 1, exceptthat the quantity of sodium chlorite was reduced from 1.5 equivalents to1.2 equivalents, (+)-valencene (1.0 g, 4.9 mmol, 1.0 equiv.) was placedin a four-neck 250-mL flask equipped with a nitrogen inlet tube,thermowell, and a mechanical stirrer. Acetonitrile (45 mL) and water (15mL) were added to the flask. N-hydroxyphthalimide (80 mg, 0.05 mmol, 0.1equiv.) was added to the solution followed by the slow addition ofsodium chlorite (80%) (0.66 g, 5.9 mmol, 1.2 equiv.) The reaction washeated to 50° C. for 18 h; it was then cooled and poured into a 10%solution of sodium sulfite (100 mL). This solution was then extractedwith diethyl ether (100 mL). The organic layer was washed with saturatedsodium bicarbonate (50 mL), dried over sodium sulfate, and absorbed ontosilica gel (2 g). This material was then purified on a silica gel column(20 g) and eluted with 5% ethyl acetate in heptane (250 mL) and 10%ethyl acetate in heptane (250 mL). The fractions containing the desiredproduct were combined and concentrated under reduced pressure to givethe desired product in 52% yield (0.37%).

Example 7 Oxidation of Premnaspirodiene to 5-Epi-β-Vetivone

To verify these conditions for premnaspirodiene, a larger scale reactionwas carried out as follows: (−)-Premnaspirodiene (distilled fromvegetable oil, ˜90% purity) (1.0 g, 4.6 mmol, 1.0 equiv.) was placed ina four-neck 250 mL flask equipped with a nitrogen inlet tube,thermowell, and a mechanical stirrer. Acetonitrile (45 mL) and water (15mL) were added to the flask. A 70% solution in water of t-butylhydroperoxide (2.94 g, 23 mmol, 5 equiv.) was added to the solutionfollowed by the slow addition of sodium chlorite (80%) (0.63 g, 5.5mmol, 1.2 equiv.). The reaction was heated to 50° C. for 18 h. Verylittle conversion of starting material was seen at this point, asdetermined by gas chromatography, likely due to the poor solubility inacetonitrile/water. t-Butanol was added (15 mL) and the homogenousreaction was heated for an additional 16 h at 50° C. The reaction wasthen cooled and poured into a 10% solution of sodium sulfite (100 mL).The solution was then extracted with diethyl ether (100 mL). The organiclayer was washed with saturated sodium bicarbonate (50 mL), dried oversodium sulfate, and adsorbed onto silica gel. The material was purifiedon a silica gel column (20 g) and eluted with 5% ethyl acetate inheptane (250 mL) and 10% ethyl acetate in heptane (250 mL). Fractionscontaining (−)-solavetivone, as determined by gas chromatography andconfirmed by nuclear magnetic resonance (NMR), were combined andconcentrated to give the desired product. The yield was 18%.

Example 8 Distillation of Premnaspirodiene

As described above, engineered yeast strains expressing the Hyoscyamusmuticus premnaspirodiene synthase gene were grown in fermentorscontaining vegetable oil (soybean oil purchased from local groceries).The oil layers were collected and pooled from numerous fermentations andsubjected to distillation under the conditions described below.

Distillation was carried out in a Pope still (wiped-film) at 100° C. anda pressure of 350 mTorr. Recovery was 4.05 g (3.96% of a ˜4% solution).Analysis by gas chromatography of the oil prior to distillation and ofthe purified premnaspirodiene distilled from the oil indicated that thedistilled product was very similar to the product contained in the oil.Recovery was virtually 100% as indicated by a lack of the detectablepremnaspirodiene in the vegetable oil following distillation. The purityof the distilled premnaspirodiene was estimated to be 92%.

The gas chromatography (GC) trace of premnaspirodiene in vegetable oilbefore distillation is shown in FIG. 7. FIG. 7A shows the graph onnormal scale. FIG. 7B expands the scale to depict the impurities presentin the premnaspirodiene before distillation. FIG. 8 provides a graphshowing the GC trace of purified premnaspirodiene after distillation.FIG. 8A shows the graph on a normal scale. FIG. 8B expands the scale todepict the lack of impurities present in the premnaspirodiene afterdistillation.

Example 9 Evaluation of 5-epi-β-vetivone

The intensity and longevity of a fragrance is based on theconcentration, intensity and longevity of the aromatic compounds used.

The performance of 5-epi-β-vetivone (EBV) as a fragrance was evaluated.EBV was compared to Nootkatone (CAS: 4674-50-4) daily over a period ofthirty days. The evaluation mixture contained a 10% solution of eachmolecule in Isopropyl Myristate (CAS: 110-27-0). The evaluation wasperformed by three trained perfumers.

The results of the evaluation are listed in Table 2. Each compound wasrated on a scale of 1 (no detectable odor) to 10 (strength of the 10%Nootkatone solution on Day 1). The initial strength of EBV was perceivedas roughly “half as intense” as Nootkatone. While EBV was noticeablyweaker then Nootkatone, it was still detectable after approximatelythree weeks. However, each compound diminished in strength after threeweeks, and both compounds exhibited similar degradation. EBV remainedproportionally weaker than Nootkatone.

TABLE 2 Nootkatone Pure (>98%) 5-epi-β-vetivone (95.1%) StrengthStrength Day 0 10 6 Day 7 9 5 Day 14 7 3 Day 21 5 2 Day 28 4 2

A fragrance can be classified or described according to the elements ofits character. The characters of both EBV and Nootkatone was describedas grapefruit, woody, and vetivert. Close and repeated evaluation of thetwo compounds indicated that EBV is similar, but slightly more “woody”in character than Nootkatone, which is more “grapefruit” in character.As such, EBV has a better, more desirable smell then currently availablecompounds. Perfumers found the character of both compounds to changerelatively little over a three week period. Additionally, EBV was foundto be moderately stable. The results indicate that EBV has along-lasting, woody, vetiver note that is not bound by natural orbotanical supply constraints and introduces little or no potential forcolor problem.

REFERENCES

The following references are cited in the specification and examples byreference number; all of these references are incorporated by thisreference in the application in their entirety.

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ADVANTAGES OF THE INVENTION

The present invention provides novel and efficient methods for theproduction and isolation of 5-epi-β-vetivone,2-isopropyl-6,10-dimethyl-spiro[4.5]deca-2,6-dien-8-one and2-isopropyl-6,10-dimethyl-spiro[4.5]deca-1,6-dien-8-one, which areuseful for their fragrant properties. These methods use premnaspirodieneas a starting material, and the present invention also provides noveland efficient methods for the production of premnaspirodiene from areadily-available terpene substrate.

The present invention provides highly purified 5-epi-β-vetivone,2-isopropyl-6,10-dimethyl-spiro[4.5]deca-2,6-dien-8-one and2-isopropyl-6,10-dimethyl-spiro[4.5]deca-1,6-dien-8-one in high yield,requiring a limited number of reaction steps and using inexpensive andreadily available starting materials.

The present invention possesses industrial applicability because theproducts, namely 5-epi-β-vetivone,2-isopropyl-6,10-dimethyl-spiro[4.5]deca-2,6-dien-8-one and2-isopropyl-6,10-dimethyl-spiro[4.5]deca-1,6-dien-8-one, are capable ofindustrial use and application as fragrances and for other purposes.

With respect to ranges of values, the invention encompasses eachintervening value between the upper and lower limits of the range to atleast a tenth of the lower limit's unit, unless the context clearlyindicates otherwise. Moreover, the invention encompasses any otherstated intervening values and ranges including either or both of theupper and lower limits of the range, unless specifically excluded fromthe stated range.

Unless defined otherwise, the meanings of all technical and scientificterms used herein are those commonly understood by one of ordinary skillin the art to which this invention belongs. One of ordinary skill in theart will also appreciate that any methods and materials similar orequivalent to those described herein can also be used to practice ortest this invention.

The publications and patents discussed herein are provided solely fortheir disclosure prior to the filing date of the present application.Nothing herein is to be construed as an admission that the presentinvention is not entitled to antedate such publication by virtue ofprior invention. Further the dates of publication provided may bedifferent from the actual publication dates which may need to beindependently confirmed.

All the publications cited are incorporated herein by reference in theirentireties, including all published patents, patent applications,literature references, as well as those publications that have beenincorporated in those published documents. However, to the extent thatany publication incorporated herein by reference refers to informationto be published, applicants do not admit that any such informationpublished after the filing date of this application to be prior art.Similarly, all GenBank sequences and other sequence informationobtainable from publicly accessible databases are herein incorporated byreference as if each was specifically and individually indicated to beincorporated by reference.

As used in this specification and in the appended claims, the singularforms include the plural forms. For example the terms “a,” “an,” and“the” include plural references unless the content clearly dictatesotherwise. Additionally, the term “at least” preceding a series ofelements is to be understood as referring to every element in theseries. The inventions illustratively described herein can suitably bepracticed in the absence of any element or elements, limitation orlimitations, not specifically disclosed herein. Thus, for example, theterms “comprising,” “including,” “containing,” etc. shall be readexpansively and without limitation. Additionally, the terms andexpressions employed herein have been used as terms of description andnot of limitation, and there is no intention in the use of such termsand expressions of excluding any equivalents of the future shown anddescribed or any portion thereof, and it is recognized that variousmodifications are possible within the scope of the invention claimed.Thus, it should be understood that although the present invention hasbeen specifically disclosed by preferred embodiments and optionalfeatures, modification and variation of the inventions herein disclosedcan be resorted by those skilled in the art, and that such modificationsand variations are considered to be within the scope of the inventionsdisclosed herein. The inventions have been described broadly andgenerically herein. Each of the narrower species and subgenericgroupings falling within the scope of the generic disclosure also formpart of these inventions. This includes the generic description of eachinvention with a proviso or negative limitation removing any subjectmatter from the genus, regardless of whether or not the excisedmaterials specifically resided therein. In addition, where features oraspects of an invention are described in terms of a Markush group, thoseschooled in the art will recognize that the invention is also therebydescribed in terms of any individual member or subgroup of members ofthe Markush group. It is also to be understood that the abovedescription is intended to be illustrative and not restrictive. Manyembodiments will be apparent to those of in the art upon reviewing theabove description. The scope of the invention should therefore, bedetermined not with reference to the above description, but shouldinstead be determined with reference to the appended claims, along withthe full scope of equivalents to which such claims are entitled. Thoseskilled in the art will recognize, or will be able to ascertain using nomore than routine experimentation, many equivalents to the specificembodiments of the invention described. Such equivalents are intended tobe encompassed by the following claims.

We claim:
 1. A fragrance composition, comprising: a compound having the structure:

in an amount effective to impart fragrance, wherein the composition is free of beta-vetivone.
 2. The fragrance composition of claim 1, wherein the compound is present in an amount of at least 0.01% by weight.
 3. The fragrance composition of claim 1 which comprises an amount of the compound of Formula (1) effective to impart fragrance in combination with conventional fragrance ingredients.
 4. A perfumed product, comprising a compound having the structure of Formula (1):

free of beta-vetivone.
 5. The perfumed product of claim 4, wherein the product is a household product, laundry product, body care product or cosmetic product.
 6. The perfumed product of claim 5, wherein the household product is a solid or liquid detergent, a fabric softener, an air freshener, a fabric freshener, an ironing water, a paper, a wipe or a bleach.
 7. The perfumed product of claim 5, wherein the body care product is a perfume, a cologne or after-shave lotion, a perfumed soap, a shower or bath salt, mousse, oil or gel, a hygiene product, a hair care product, a shampoo, a deodorant or antiperspirant.
 8. A perfuming composition, comprising a compound of Formula (1):

in an amount sufficient to give a fragrance to the composition, wherein the composition is free of beta-vetivone.
 9. The perfuming composition of claim 8 that comprises at least one perfumery carrier, perfumery base, or perfumery adjuvant.
 10. A fragrance application, comprising a compound of Formula (1):

wherein the application is free of beta-vetivone.
 11. The fragrance application of claim 10 wherein the fragrance application is a household product, laundry product, body care product or cosmetic product.
 12. The fragrance application of claim 11, wherein the household product is a solid or liquid detergent, a fabric softener, an air freshener, a fabric refresher, an ironing water, a paper, a wipe or a bleach.
 13. The fragrance application of claim 11, wherein the body care product is a perfume, a cologne or after-shave lotion, a perfumed soap, a shower or bath salt, mousse, oil or gel, a hygiene product, a hair care product, a shampoo, a deodorant or antiperspirant.
 14. A method of imparting a woody, grapefruit, or vetivert odor to a fragrance, comprising providing a compound of Formula (1):

free of beta-vetivone to the fragrance.
 15. A method of imparting a woody, grapefruit, or vetivert odor to a fragrance composition, comprising providing a compound of Formula (1):

free of beta-vetivone to the fragrance composition.
 16. A method of imparting, improving, enhancing or modifying a fragrance formulation, comprising adding an olfactory acceptable amount of a compound of Formula (1):

free of beta-vetivone to the fragrance formulation.
 17. The method of claim 16, wherein the olfactory acceptable amount is from about 0.005 to about 10 weight percent of the fragrance formulation.
 18. The method of claim 17, wherein the olfactory acceptable amount is from about 0.5 to about 8 weight percent of the fragrance formulation.
 19. The method of claim 18, wherein the olfactory acceptable amount is from about 1 to about 7 weight percent of the fragrance formulation.
 20. A mixture, comprising: a compound of Formula (1):

free of beta-vetivone; and an auxiliary ingredient compatible with the compound of Formula (1), wherein the weight ratio of the compound of Formula (1) is in the range of from about 1:1 up to about 1:5.
 21. A fragrance modifying composition, comprising: a compound of Formula (1):

free of beta-vetivone; and an auxiliary ingredient compatible with the compound of Formula (1), wherein the weight ratio of the compound of Formula (1) is in the range of from about 1:1 up to about 1:5.
 22. A perfume composition, comprising: a compound of Formula (1):

free of beta-vetivone; and at least one compatible adjuvant, wherein the ratio of the compound of Formula (1) is in the range of from about 1:1 up to about 1:5.
 23. A cologne composition, comprising: a compound of Formula (1):

free of beta-vetivone; and at least one compatible adjuvant, wherein the ratio of the compound of Formula (1) is in the range of from about 1:1 up to about 1:5.
 24. A method of formulating a perfumed product, comprising incorporating into at least one compatible perfumery carrier, perfumery base, or perfumery adjuvant a compound of Formula (1):

free of beta-vetivone.
 25. The method of claim 24, further comprising an auxiliary ingredient compatible with the compound of Formula (1).
 26. A compound of Formula (1):

that is isolated, purified, and free of beta-vetivone.
 27. A compound of Formula (1):

that is isolated, purified, and free of beta-vetivone that is produced by the process of: (a) subjecting (−)-premnaspirodiene to oxidation of an allylic carbon to form (−)-solavetivone; and (b) subjecting the (−)-solavetivone to acid-catalyzed isomerization to form 5-epi-beta-vetivone; wherein time and temperature conditions for step (b) are such that the predominant product is 5-epi-beta-vetivone.
 28. A compound of Formula (1):

that is isolated, purified, and free of beta-vetivone and that is produced by the process of: (a) providing a terpene substrate to a host cell transformed or transfected with a vector comprising a sequence encoding a Hyoscyamus muticus premnaspirodiene synthase gene; (b) culturing the host cell under conditions suitable to produce (−)-premnaspirodiene from the terpene substrate; (c) isolating the (−)-premnaspirodiene; (d) subjecting the isolated (−)-premnaspirodiene to oxidation of an allylic carbon to form (−)-solavetivone; and (e) subjecting the (−)-solavetivone formed in step (d) to acid-catalyzed isomerization to form 5-epi-beta-vetivone; wherein time and temperature conditions for step (e) are such that the predominant product is 5-epi-beta-vetivone. 